*Round1 RT Primers (randN): 5'-/5Phos/
*Round2 Ligation Oligos: 5'-/5Phos/
*Round3 Ligation Oligos: 5'- AGAA
Round 2 barcode linker (SB83): 5'-
Round 3 barcode linker (SB80): 5'-
Round 3 blocking strand 1 (SB81): 5'- GCTTCGCTGCAATCGGACCTCGATTGCA -3'
Round 3 blocking strand 2 (SB82): 5'-
Round 2 blocking strand 1 (SB84): 5'- GCCAGASACGTTAGGCAGGACCTAACGT -3'
Round 2 blocking strand 2 (SB85): 5'-
Nextera Tn5 binding site (19-bp Mosaic End (ME)): 5'-
Nextera N/S5xx primer entry point (s5): 5'-
Nextera N7xx primer entry point (s7): 5'-
NEB i50x primer (NEB kit E7600): 5'-
Nextera (XT) N7xx Index primer: 5'-
Illumina TruSeq Read 1 primer: 5'-
Index 1 sequencing primer (i7): 5'-
Index 2 sequencing primer (i5): 5'-
Illumina Nextera Read 2 primer: 5'-
Illumina P5 adapter: 5'-
Illumina P7 adapter: 5'-
*NOTE: The first nt of the
Anneal Round2 Ligation Oligos with Round 2 barcode linker (SB83): 5'-/5Phos/ GCTTCGC [7-bp Round2 barcode] CCTCCTAC 3'-WGGAGGATG CGGTCTS -5' Anneal Round3 Ligation Oligos with Round 3 barcode linker (SB80): 5'- AGAATACACGACGCTCTTCCGATCT [7-bp UMI] [7-bp Round3 barcode] GGTCCTTG 3'-WCCAGGAACCGAAGCG -5'
5'-/5Phos/ GCCAGA [7-bp Round1 barcode] NNNNNN---> 3'- XXXXXXX...XXXXXXX -5'
5'-/5Phos/ GCTTCGC [7-bp Round2 barcode] CCTCCTAC GCCAGA [7-bp Round1 barcode] NNNNNN---> 3'-WGGAGGATG CGGTCTS -5' 3'- XXXXXXX...XXXXXXX -5'
5'- AGAA TACACGACGCTCTTCCGATCT [7-bp UMI] [7-bp Round3 barcode] GGTCCTTGGCTTCGC [7-bp Round2 barcode] CCTCCTAC GCCAGA [7-bp Round1 barcode] NNNNNN---> 3'-WCCAGGAACCGAAGCG -5' 3'-WGGAGGATG CGGTCTS -5' 3'- XXXXXXX...XXXXXXX -5'
5'- AGAA TACACGACGCTCTTCCGATCT [7-bp UMI] [7-bp Round3 barcode] GGTCCTTGGCTTCGC [7-bp Round2 barcode] CCTCCTAC GCCAGA [7-bp Round1 barcode] XXX.XXX -3' 3'- TCTTATGTGCTGCGAGAAGGCTAGA [7-bp UMI] [7-bp Round3 barcode] CCAGGAACCGAAGCG [7-bp Round2 barcode] GGAGGATG CGGTCT [7-bp Round1 barcode] XXX.XXX -5'
Product 1 (s5 at both ends, not amplifiable due to PCR primers used, see the next step): 5'- TCGTCGGCAGCGTC AGATGTGTATAAGAGACAG XXXXXXXXXXXX...XXXCTGTCTCTTATACACATCT TCTACACATATTCTCTGTC XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGA CTGCGACGGCTGCT -5' Product 2 (s7 at both ends, not amplifiable due to PCR primers used, see the next step): 5'-GTCTCGTGGGCTCGG AGATGTGTATAAGAGACAG XXXXXXXXXXXX...XXXCTGTCTCTTATACACATCT TCTACACATATTCTCTGTC XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG -5' Product 3 (different s5 and s7 at both ends, not amplifiable, due to PCR primers used, see the next step): 5'-TCGTCGGCAGCGTC AGATGTGTATAAGAGACAG XXXXXXXXXXXX...XXXCTGTCTCTTATACACATCT TCTACACATATTCTCTGTC XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG -5' Product 4 (s5 at one end, 3' of cDNA at the other end, not amplifiable due to PCR primers used, see the next step): 5'- AGAATACACGACGCTCTTCCGATCT [7-bp UMI] [7-bp Round3 barcode] GGTCCTTGGCTTCGC [7-bp Round2 barcode] CCTCCTAC GCCAGA [7-bp Round1 barcode] XXX.XXXCTGTCTCTTATACACATCT -3' 3'- TCTTATGTGCTGCGAGAAGGCTAGA [7-bp UMI] [7-bp Round3 barcode] CCAGGAACCGAAGCG [7-bp Round2 barcode] GGAGGATG CGGTCT [7-bp Round1 barcode] XXX.XXXXXXXXXXXXGACAGAGAATATGTGTAGA CTGCGACGGCTGCT -5' Product 5 (s7 at one end, 3' of cDNA at the other end, the only amplifiable fragment, see the next step): 5'- AGAATACACGACGCTCTTCCGATCT [7-bp UMI] [7-bp Round3 barcode] GGTCCTTGGCTTCGC [7-bp Round2 barcode] CCTCCTAC GCCAGA [7-bp Round1 barcode] XXX.XXXCTGTCTCTTATACACATCT -3' 3'- TCTTATGTGCTGCGAGAAGGCTAGA [7-bp UMI] [7-bp Round3 barcode] CCAGGAACCGAAGCG [7-bp Round2 barcode] GGAGGATG CGGTCT [7-bp Round1 barcode] XXX.XXXXXXXXXXXXGACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG -5'
5'- AATGATACGGCGACCACCGAGATCTACAC [i5]ACACTCTTTCCCTACACGACGCTCTTCCGATCT --------> 5'- AGAATACACGACGCTCTTCCGATCT [7-bp UMI] [7-bp Round3 barcode] GGTCCTTGGCTTCGC [7-bp Round2 barcode] CCTCCTAC GCCAGA [7-bp Round1 barcode] XXX.XXXCTGTCTCTTATACACATCT -3' 3'- TCTTATGTGCTGCGAGAAGGCTAGA [7-bp UMI] [7-bp Round3 barcode] CCAGGAACCGAAGCG [7-bp Round2 barcode] GGAGGATG CGGTCT [7-bp Round1 barcode] XXX.XXXXXXXXXXXXGACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG -5' <----GGCTCGGGTGCTCTG [i7]TAGAGCATACGGCAGAAGACGAAC -5'
5'- AATGATACGGCGACCACCGAGATCTACAC NNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATCT NNNNNNN NNNNNNN GGTCCTTGGCTTCGC NNNNNNN CCTCCTAC GCCAGA NNNNNNN XXX.XXXCTGTCTCTTATACACATCT CCGAGCCCACGAGAC NNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3' 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG NNNNNNNNTGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA NNNNNNN NNNNNNN CCAGGAACCGAAGCG NNNNNNN GGAGGATG CGGTCT NNNNNNN XXX.XXXGACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG NNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'Illumina P5 8bp i5TruSeq Read 1 7-bp 7-bp Round3 7-bp Round2 7-bp cDNAME s7 8bp i7Illumina P7 UMI Round3 linker Round2 linker Round1 barcode barcode barcode
5'- ACACTCTTTCCCTACACGACGCTCTTCCGATCT --------------------------------------------------------> 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG NNNNNNNNTGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA NNNNNNN NNNNNNN CCAGGAACCGAAGCG NNNNNNN GGAGGATG CGGTCT NNNNNNN XXX.XXXGACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG NNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
5'- CTGTCTCTTATACACATCT CCGAGCCCACGAGAC -------> 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG NNNNNNNNTGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA NNNNNNN NNNNNNN CCAGGAACCGAAGCG NNNNNNN GGAGGATG CGGTCT NNNNNNN XXX.XXXGACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG NNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
5'- AATGATACGGCGACCACCGAGATCTACAC NNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATCT NNNNNNN NNNNNNN GGTCCTTGGCTTCGC NNNNNNN CCTCCTAC GCCAGA NNNNNNN XXX.XXXCTGTCTCTTATACACATCT CCGAGCCCACGAGAC NNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3' <-------TGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA
5'- AATGATACGGCGACCACCGAGATCTACAC NNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATCT NNNNNNN NNNNNNN GGTCCTTGGCTTCGC NNNNNNN CCTCCTAC GCCAGA NNNNNNN XXX.XXXCTGTCTCTTATACACATCT CCGAGCCCACGAGAC NNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3' <------GACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG -5'